The generation or destruction of tissue requires constant reorganization and restructuring of the extracellular matrix (ECM) components including interstitial collagens, basement membrane collagen, fibronectin, laminin, aggrecan, and various proteoglycans. Heinegard and Oldberg, FASEB J. 1989, 3, 2042-2051; Woessner FASEB J. 1991, 5, 214-2154. Normal types of remodeling processes include embryonic development, post-partum involution of the uterus, ovulation, wound healing, and bone and growth plate remodeling. Woessner et al. Steroids 1989, 54, 491-499; Weeks et al. Biochim Biophys Acta 1976, 445, 205-214; Lepage and Gache EMBO J. 1990, 9, 3003-3012; Wride and Sanders Dev-Dyh. 1993, 198(3) 225-39. Similar processes also occur in disease states such as joint destruction in rheumatoid and osteoarthritis, periodontia and tumor cell metastasis. Thompson and Oegema J Bone Joint Surg. 1979, 61, 407-16; Reynolds et al. Adv-Dent-Res. 1994, 8(2) 312-9. One example of these processes is the migration of macrophages to the site of inflammation as in the case of synovial tissue in rheumatoid arthritis. Cutolo et al. Clin. and Exper. Rheum. 1993, 11, 331-339. The ECM components are regulated, in both normal and disease states, by various exogenous and endogenous factors. For example, in tumor formation, the differentiation state of the cell can increase the rate of degradation of the ECM. Benya Pathol. Immunopathol. Res. 1988, 7, 51-54. Likewise, the presence of metalloproteinases or their inhibitors can alter the composition of the ECM. An imbalance of metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMP) has been shown to contribute to the pathogenesis of osteoarthritis. Dean et al. J. Clin. Invest. 1989, 84: 678-685. Cytokines, growth factors, and the extracellular environment can all contribute to the alteration of the ECM. Tyler Biochem J. 1985, 227, 869-878; Dinarello Sem Immunol. 1992, 4, 133-145; McConnell et al. J. Cell Biol. 1987, 105, 1087-98.
The growth of cartilage and bone is actualized by cells such as articular chondrocytes and osteoblasts. The main function of these cells in immature tissue is the deposition and remodeling of the cartilage or bone matrix. In adult tissue, these cells maintain this matrix in order to ensure its proper function. In both cases, this encompasses secretion of the extracellular components as well as secretion of proteins involved in the turnover of the ECM.
A major species of protein secreted by these cells and involved in the turnover of the ECM are the metalloproteinases. Woessner FASEB J. 1991, 5, 214-2154. A new type of secretory glycoprotein has also been identified in human cartilage, osteoblasts, synovial cells, sheep and bovine oviduct and mammary cells, and macrophages. Nyrikos and Golds Biochem J. 1990, 268, 265-268; Hakala et al. J. Biol. Chem. 1993, 268(34) 25803-25810; Johansen et al. J. Bone and Min. Res. 1992, 7(5) 501-511; Rejman and Hurley Biochem Biophys. Res. Commun. 1988, 150, 329-334; DeSouza and Murray Endocrinology 1995, 136(6) 2485-2496; Hollak et al. J. Clin. Invest. 1994, 93, 1288-92; Arias et al. Biol. of Reproduction 1994, 51, 685-694. These novel mammalian proteins all share regions of significant homology to the bacterial and fungal chitinases and, therefore, are referred to herein as xe2x80x9cchitinase-likexe2x80x9d proteins. Chitinases are enzymes that hydrolyze glycosidic bonds. They bear a subtle similarity to lysozymes from mammals and function as endoglycosidases with a specificity for N-acetyl-glucosamine linkages. However, these types of chitin-like structures, homopolymers of N-acetyl-glucosamine, are not normally encountered in mammalian tissue.
The human cartilage glycoprotein, HC gp-39, is a protein with an apparent molecular weight of approximately 39 kDa secreted by both articular chondrocytes and synovial fibroblasts. Nyrikos and Golds Biochem J. 1990, 268, 265-268; Hakala et al. J. Biol. Chem. 1993, 268(34), 25803-25810. This protein has been described as a marker for joint injury, appearing in the blood and synovial fluid from patients diagnosed with rheumatoid arthritis. Johansen et al. British J. of Rheumatology 1993, 32, 949-955. The gene encoding this protein has been cloned and is expressed specifically in cartilage and synovial cells of rheumatic joints. Hakala et al. J. Biol. Chem. 1993, 268(34), 25803-25810. The protein YKL-40 has also been identified as one of the major secretory products of cultured human osteoblastic cells (osteocarcinoma cell line MG-63) expressed in response to 1,25-dihydroxyvitamin D3 stimulation. Johansen et al. J. Bone and Min. Res. 1992, 7(5), 501-511; Johansen et al. Br. J. Rheumatol. 1993, 32, 949-55. The N-terminal portion of YKL-40 was sequenced and found to be identical to HC gp-39. Upon further sequencing, YKL-40 and HC gp-39 were found to be identical.
Chitotriosidase is an enzyme which has been identified as a member of this xe2x80x9cchitinase-likexe2x80x9d family. Renkema et al. J. Biol Chem. 1995, 27C, 2198-2202; Hollak et al. J. Clin. Invest. 1994, 93, 1288-92. This protein also has an apparent molecular weight of 39 kDa and shares N-terminal homologies with HC gp-39, the bovine mammary protein, and several bacterial chitinases. Activity of this enzyme was originally detected from cells of patients afflicted with Gaucher Disease (GD). Gaucher Disease is an inherited deficiency in the activity of glucocerebrosidase, a lysosomal hydrolase. This defect results in an accumulation of glucosylceramide (glucocerebroside) in the lysosomes of macrophages. Accumulation of lipid-laden macrophages results in hepatosplenomegaly, bone lesions, and neurological anomalies. After morphological differentiation of monocytes into macrophages in culture, the cells begin to produce and secrete increasing amounts of chitotriosidase. This increase is, on average, 600 times greater in GD patients than in patients with other pathological conditions. The elevation in chitotriosidase activity can be effectively reduced, however, upon initiation of enzyme supplementation therapy. Unlike the other members of the chitinase-like family, chitotriosidase has chitolytic activity. Like the bacterial enzyme, it has the ability to degrade chitin azure, a polymer of beta-1-4-linked N-acetylglucosamine moieties.
A new lymphocyte-associated protein of the chitinase-like family, referred to as HC gp-39L, has now been identified. HC-gp39L protein is believed to be involved in tissue remodeling in the mammalian cell and thus serve as useful tools in the development of therapeutics and diagnostics for tissue remodeling disorders.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is HC gp-39L, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human HC gp-39L, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there is provided a process for producing HC gp39L by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human nucleic acid sequence of HC gp-39L, under conditions promoting expression of these proteins and subsequent recovery of these proteins.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing HC gp-39L polypeptide, for therapeutic purposes, for example, in the treatment of tissue remodeling disorders.
In accordance with yet a further aspect of the present invention, there is provided a process for targeting HC gp 39L located on the surface of lymphocytes with specific antibodies, for therapeutic purposes, such as the treatment of autoimmune or tissue remodeling disorders.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing HC gp-39L polypeptide for diagnostic purposes, for example, in the diagnosis of tissue remodeling disorders.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.